This article describes a method for purifying calcium-binding proteins using calcium-dependent hydrophobic interaction chromatography. The technique leverages the binding properties of calcium ions to enhance the separation of target proteins from non-calcium-binding proteins.
Calcium-dependent hydrophobic interaction chromatography is a technique to separate calcium-binding proteins from other non-calcium-binding proteins in a sample.
To begin, take a pre-assembled chromatography column containing a matrix of cross-linked agarose beads derivatized with hydrophobic phenyl groups to facilitate interactions with the target proteins. Equilibrate it with a binding buffer containing a very high concentration of calcium ions for maximum resin and target protein interaction during the subsequent sample loading.
Take a sample containing the dialyzed protein mixture and supplement it with an equal concentration of calcium chloride as used in the binding buffer for column equilibration. This maximizes protein binding to the column.
Calcium ions bind to specific proteins in the lysate, inducing a conformational change in these protein molecules exposing their hydrophobic regions.
Filter the mixture to separate impurities and aggregates to prevent them from clogging the column. Load this filtrate into the pre-equilibrated column. Proteins with exposed hydrophobic regions interact with the corresponding hydrophobic phenyl groups of the resin.
Wash using the salt-containing binding buffer to remove unbound proteins for enhancing the sample purity in later steps. Pass the calcium chloride-free elution buffer containing EDTA - a divalent chelator - through the column.
As EDTA chelates the calcium ions, the hydrophobic interactions between the target protein and resin are disrupted. The target proteins become mobile and elute from the column.
To continue the protein purification, after the protein dialysis, add calcium chloride to the sample to a final concentration of 25 millimolar, which will facilitate binding of S100A12 to the resin in the next step. Then, filter the sample through a filter with a pour size of 0.45 microns.
Equilibrate HIC buffers and samples to 4 degrees Celsius. Next, connect column buffers HIC-A and B and the column, to the system and adjust the parameters.
Start the method to equilibrate the column with 1 to 2 column volumes of buffer HIC-A, and load the sample, and the 'wash unbound sample block' when the UV signal reaches baseline level again.
Then, start elution with a calcium chelator-containing buffer EDTA. Collect 2 milliliters of peak fractions during elution.
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